DESCRIPTION: The specific aim of this proposed research is to understand the repair of spontaneous and double-strand-break induced DNA damage. Building on their past successes with yeast as an experimental system, these applicants use molecular genetic and biochemical approaches to study the genes involved in the repair of these lesions. Their efforts focus on genes that have been isolated or are planned to be isolated that are required for the processing of DNA damage as well as those that alter the level of spontaneous DNA damage. They will continue their focus on the repair of DNA damage during mitosis, however they will also assess the effect of mutations that they isolate on meiosis to help define the common and unique genes involved with each of these processes. The researchers will attempt to define further the biochemical activities of some of the key gene products involved in this process. Their specific approaches to these problems are: 1) To characterize further the Rfa1 recombination pathway by (a) studying Rfa1-interacting proteins, RTC1 and RTC2 as well as exploring the interaction between Rad52 and RP-A, and (b) studying new mutations that have been found to decrease Rfa1-stimulated recombination: rsp5-25 and drf 3. 2) Continue the biochemical studies on Rad52 by: (a) further characterizing the strand annealing activity of Rad52 by exploring substrate specificity, (b) determining whether Rad52 can also do strand exchange, (c) searching for mutations that disrupt the biochemical activity of Rad52 and its related biological functions and (d) identifying Rad52-interacting proteins by direct protein-protein interactions. 3) Embark on a search for proteins involved in the branch migration and/or resolution of Holliday Junctions, and 4) Explore the relationship between DNA damage and mitotic checkpoints by characterizing SML1, a suppressor of two important radiation sensitive/checkpoint mutations, rad53 and mec1. The combination of genetic and biochemical approaches to the many issues related to the repair of double strand breaks in yeast are expected by the researchers to continue to yield new insights into this important biological phenomenon.